Intracellular distribution of NADPH-cytochrome c reductase in rat hepatocytes studied by direct ferritin-immunoelectron microscopy.
NADPH-cytochrome c reductase was purified from rat liver microsomes and the monospecific antibodies to the reductase were prepared from the antiserum by affinity chromatography using immunoadsorbent gel. Ferritin was coupled to the specific antibodies and the approximately equimolar conjugates were isolated by gel filtration. By direct ferritin-immunoelectron microscopy, using these conjugates, it was revealed that the ferritin particles are localized exclusively on the microsomal vesicles and the outer nuclear envelope. In contrast, binding of ferritin particles to Golgi membranes, outer mitochondrial membranes and plasma membranes was slight and at control level. On each microsomal vesicle, the ferritin particles were distributed heterogeneously, sometimes forming clusters. An assay of the binding of equimolar conjugates with microsomes showed that microsomes bind approximately 1 mol of antibody per mol of reductase. From these data the maximum number of ferritin particles that can bind with microsomes was calculated. This number is in agreement with the average number of ferritin particles bound per microsome, as determined experimentally by observing a number of cross-sectional profiles of microsomal vesicles previously incubated with the conjugates at saturation level. This showed that the distribution of the reductase could be analysed semi-quantitatively by the present ferritin-immunoelectron-microscopical analyses. It was also shown that smooth microsomes can bind more conjugates than rough microsomes. The average number of ferritin particles on each microsomal vesicle increased in proportion to the increase in the amount of reductase in the microsomes after treatment with phenobarbital. Finally, the non-random distribution of ferritin particles on microsomal vesicles was confirmed by statistical analysis of electron micrographs of a number of the labelled microsomes.
D000818:Animals; D005293:Ferritins; D006056:Golgi Apparatus / Q000201:enzymology / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000201:enzymology / Q000648:ultrastructure; D008297:Male; D008854:Microscopy, Electron; D008862:Microsomes, Liver / Q000201:enzymology*; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013347:Subcellular Fractions / Q000201:enzymology / Q000648:ultrastructure
D005293:Ferritins; D009251:NADPH-Ferrihemoprotein Reductase